7. Project Summary/Abstract Deficiencies in complex I of the mitochondrial electron transport chain represent an important class of human mitochondrial diseases that include Leigh Syndrome, Leber?s hereditary optic nephropathy, neonatal lactic acidosis, cardiomyopathy, and encephalopathy. Leigh Syndrome is generally regarded as the most common mitochondrial disease of infancy and is characterized primarily by severe neurological defects. There is currently no effective treatment. A mouse model has been developed in which the complex I structural subunit NDUFS4 is knocked-out. Ndufs4-/- mice show a profound neurodegenerative phenotype including retarded growth rate, lethargy, loss of motor skill, blindness, weight loss, and early death. These symptoms are recapitulated in human patients with Ndufs4 mutations, as well as other Leigh Syndrome-associated mutations. We have recently reported that treating Ndufs4-/- mice with the drug rapamycin, a specific inhibitor of the mammalian target of rapamycin complex 1 (mTORC1), results in two to three-fold increase in survival, dramatic attenuation of neurologial defecits, and reduced neurodegeneration. In unpublished studies, we have observed similar effects from the NAD+ precursor nicotinamide mononucleotide (NMN) -glucosidase inhibitor acarbose. The broader goals this proposal to define the mechanistic basis for suppression of mitochondrial disease by rapamycin, NMN, and acarbose in the Ndufs4 knockout (KO) mouse. This will be accomplished broadly by assessing changes in the metabolome and phosphoproteome, and specifically by determining the effects of these interventions on mitochondrial function, mTOR signaling, NAD homeostasis, and activity of the NAD-dependent mitochondrial SIRT3 deacetylase. We will test the specific hypothesis that these intervention act by enhancing SIRT3 activity by creating animals lacking both NDUFS4 and SIRT3 and determining whether this blocks or attenuates rescue of the disease. These studies promise to enhance our understanding of mitochondrial function in the context of complex I deficiency and facilitate the development of interventional strategies to improve the lives of patients with mitochondrial disease.